The present invention relates to a method for preventing the unfolding of a (poly)peptide during drying and/or inducing the (re-)folding of a (poly)peptide after drying, comprising the step of embedding the (poly)peptide in an aqueous solution, wherein the solution comprises (i) at least three different amino acids; or (ii) at least one dipeptide or tripeptide; and wherein the solution is free or substantially free of (a) sugar; and (b-i) protein; and/or (b-ii) denaturing compounds; and (c) silanes.
In this specification, a number of documents including patent applications and manufacturer's manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
Proteins are large macromolecules made up of a sequence of amino acids and characterized by a unique 3D structure corresponding to their biologically active state. The native structure of a protein molecule is the result of a fine balance among various interactions including covalent linkages, hydrophobic interactions, electrostatic interactions (charge repulsion and ion pairing=salt bridge), hydrogen bonding and van der Waals forces. Among these forces, hydrophobic interactions seem to be the dominant forces. Intra-protein and protein-solvent interactions both play an important role in maintaining the protein structure and its stability. Proteins are generally not very stable, as the stabilization energy of the native state is mostly between 5 and 20 kcal/mol, which is equivalent to that of a few hydrogen bonds. Since the folded state is only marginally more stable than the unfolded state, any change in the protein environment may trigger protein degradation or inactivation.
Protein stability is a result of balancing between destabilizing and stabilizing forces. The destabilizing forces are mainly due to the large increase in entropy of unfolding, and the stabilizing forces are provided by a few non-covalent interactions. Disruption of any of these interactions will shift the balance and destabilize a protein and many factors are known that disrupt this delicate balance and affect protein stability. These include for example temperature, pH, ionic strength, metal ions, surface adsorption, shearing, shaking, additives, solvents, protein concentration, purity, morphism, pressure, and freeze/thawing-drying. Chemical transformations that can lead to protein instability include e.g. deamidation, oxidation, hydrolysis, isomerization, succinimidation, disulfide bond formation or breakage, non-disulfide crosslinking, and deglycosylation. One of the most challenging tasks in the development of protein pharmaceuticals therefore is to deal with their physical and chemical instabilities and to provide a formulation able to stabilize the protein in order to achieve an acceptable shelf life.
A common phenomenon of protein instability is formation of protein aggregates that can be soluble or insoluble, chemical or physical, reversible or irreversible. Under certain conditions (or sometimes with time/shelf life), the secondary, tertiary, and quaternary structure of a protein may change and lead to protein unfolding and/or the subsequent formation of protein aggregates. Aggregation of proteins is rapidly emerging as a key issue underlying multiple deleterious effects for protein-based therapeutics, including loss of efficacy, bioavailability, stability and mobilization of an unwanted host immune response toward the protein therapeutic. In particular, antibodies against therapeutic proteins can develop during treatment with a therapeutic protein, which might neutralize or otherwise compromise the clinical effects of these therapeutic proteins and can also be associated with serious side effects such as e.g. cross-reactivity with autologous proteins. The presence of any aggregates in a protein pharmaceutical is therefore generally not acceptable for product release. Protein aggregation may be induced by a variety of physical factors, however, protein aggregation and the rate and mechanism of protein aggregation is generally protein dependent.
The most important factor affecting protein stability is temperature. In general, the higher the temperature, the lower the protein stability. Proteins are usually stable in a certain temperature range. High temperatures cause denaturation of many protein pharmaceuticals. Although protein denaturation at high temperatures can be reversible depending on experimental conditions, high temperatures also accelerate chemical degradation, such as increased hydrolysis of aspartate residues, deamidation of asparagine or glutamine residues. Most importantly, degradation mechanisms in proteins often change depending on the temperature, which is of particular relevance for example during spray-drying or storage and transport, where the cooling-chain may become interrupted.
Furthermore, proteins are often only stable in a narrow pH range and the rate of protein aggregation can be strongly affected by pH. Formulation pH, like temperature, may affect both physical and chemical stability of proteins. Different chemical degradations may be facilitated at different pHs. This explains why degradation products are different at different pHs for the same protein. Hydrolysis can easily occur at aspartate residues under mild acidic conditions. Deamidation of asparagine and glutamine residues readily takes place under strongly acidic, neutral, and basic conditions. Under basic conditions, many reactions can occur, such as peptide bond hydrolysis, deamidation, hydrolysis of arginine to ornithine, β-elimination and racemization, and double bond formation. The pH effect on chemical stability can further be altered in the presence of excipient.
Proteins can also be adsorbed to many surfaces and interfaces, such as container surfaces, ice/water interfaces and air/water interfaces. Protein adsorption in air/water interfaces starts with the creation of an area for anchoring the protein molecule, followed by subsequent reorientation and rearrangement of the adsorbed molecules at the interface. The severity of adsorption is protein-dependent and does not seem to depend on size and pl of proteins. The secondary structure of a protein, such as IgG, may change significantly at such an adsorption surface. Therefore, surface adsorption may result in loss and/or destabilization of a protein.
Protein surface adsorption is usually concentration-dependent and may reach a maximum—at least for certain proteins—above certain protein concentrations. The type (i.e. material) of a container or membrane employed for e.g. sterile filtration, dialysis or concentration of the protein has a significant influence on protein adsorption to the surface. Protein surface adsorption is further of particular relevance for example during the drying processes like freeze-drying or spray-drying.
The handling of proteins also affects stability. For example, proteins can be denatured due to shaking or shearing. Shaking, such as employed during reconstitution of dried samples, can create a hydrophobic air/water or air/surface interface, which results in alignment of protein molecules at the interface, leading to unfolding of the protein to maximize exposure of hydrophobic residues to the air or surface and the initiation of aggregation. The hydrophobic surfaces that cause protein aggregation during shaking can be either gaseous or solid. Similarly, shearing, such as encountered during spray-drying or spray-freeze drying, also exposes hydrophobic areas of proteins, thereby initiating aggregation. Different proteins may tolerate shearing inactivation to differing degrees. Rigidity of the protein structure and the number of hydrophobic residues on the protein surface might contribute to such different levels of shear tolerance.
Also salts can have an effect on protein stability, although their effect is complex, partly because of the complex ionic interactions on fully exposed surfaces and in fully or partially buried interior of proteins. Salts may stabilize, destabilize, or have no effect on protein stability depending on the type and concentration of salt, nature of ionic interactions and the presence and amount of charged residues in proteins. The salt effect further strongly depends on the pH of the solution, which dictates charged state of ionizable groups.
Depending on the type and concentration, also metal ions may stabilize or destabilize a protein. Since the negative counter ions may also significantly affect protein stability either positively or negatively, contribution of metal ions to protein stability should be carefully interpreted. The number of stabilizing metal ions required for each individual protein molecule is protein-dependent, and the metal ions may or may not be mutually replaceable for protein stability. Metal ions may significantly affect protein stability without affecting much of its secondary structure. Trace amounts of metal ions in protein formulations may catalyze oxidation in proteins namely via the Fenton pathway, targeting in particular the residues methionine, cysteine, histidine, tryptophan, tyrosine, proline, arginine, lysine, or threonine. The catalysis depends on the concentration of the metal ions, and the metal-catalyzed reaction can be facilitated in the presence of a reducing agent such as ascorbat or RSH. Metal ions, oxygen, and reducing agents can generate reactive oxygen species capable of oxidizing proteins.
In close relation to metal ions are chelating agents, such as EDTA and citric acid, that may either destabilize a protein by binding to the protein and/or its critical metal ions or stabilize the protein by binding to any harmful metal ions. Since transition metal ions can catalyze protein oxidation, ion chelating agents should be able to protect a protein from metal catalyzed oxidation. In many cases, however, the effect of chelating agents is more complex. The net effect depends on the metal ions, oxidation mechanism, and the type of the chelating agent.
Furthermore, protein aggregation is generally concentration dependent. The effect of the concentration of a protein on its aggregation depends on the mechanism of aggregation and the experimental conditions. In some cases, protein concentration also affects chemical degradation to a certain degree. On the other hand, concentrated protein solutions can be more resistant against freezing-induced protein aggregation and loss of activity.
Purity of the protein preparation is another important aspect, as the presence of trace amounts of enzymes, metal ions, or other contaminants can potentially affect protein stability.
High pressure can furthermore cause protein unfolding, because the volume of protein-solvent systems is smaller in the unfolded state. In other words, unfolded proteins are more compressible than folded proteins, which may play a role during freeze-drying or spray-freeze drying.
Also many chemical reactions are responsible for inactivation of protein drugs. In many cases, several reactions can happen simultaneously in proteins, making separation and identification of protein degradation products very difficult. To prevent proteins from chemical inactivation, the dominant reaction should first be identified and inhibited. This can be achieved to a certain degree by adjusting the formulation pH away from favorable ranges. The location of labile amino acids in a protein is critical in determining their chemical reactivity. Chemical reactions of many amino acids in proteins require a certain local flexibility and thus the rate of a reaction may be higher in denatured proteins or small peptides with high flexibility than the native proteins. Native protein conformation therefore needs to be protected to prevent or inhibit potential chemical degradation.
Deamidation, which in many cases is the major degradation pathway in proteins, also appears to be the most common degradation pathway in protein pharmaceuticals. The two amino acids susceptible to deamidation in proteins are asparagine and glutamine, whereas asparagine is the more labile amino acid. Deamidation of asparagine in proteins and peptides in an aqueous solution can proceed at much higher rate than hydrolysis of a peptide bond. The rate, mechanism, and location of deamidation in proteins are pH dependent. Furthermore, the relative positions of asparagine and/or glutamine in proteins affects their relative rate of deamidation, as do neighboring amino acids at deamidation sites in proteins. The most labile sequence seems to be Asn-Gly and the rate of deamidation in proteins is further influenced by secondary structure of proteins.
Another chemical reaction that can result in the inactivation of proteins is oxidation. In particular, the side chains of histidine, cysteine, tryptophan and tyrosine residues are potential sites of oxidation. Oxidation at these sites can be catalyzed by trace amount of transition metal ions (site-specific process) or enhanced by oxidants or upon exposure to light (non-site specific process). The site specificity is due to generation of and oxidation by reactive oxygen species at specific metal-binding sights. The most easily oxidizable sites are the thio groups on methionine and cysteine. Methionine residues in proteins can be easily oxidized by atmospheric oxygen. The formulation pH may affect the rate of oxidation by changing the oxidation potential of oxidants, the affinity of binding between catalytic metal ions and the ionizable amino acids, and the stability of oxidation intermediates. Moreover, exposure of proteins to ionizing radiation like gamma- or electron beam radiation may result in oxidation of amino acid side chains in the protein by the generated reactive oxygen species.
Disulfide bonds are often critical in controlling both protein activity and stability. Free cysteine residues in proteins can be oxidized easily to form disulfide bond linkages or cause thio-disulfide exchanges, causing protein aggregation or polymerization. Thio-disulfide exchange in a protein is a reaction between an ionized thiol group (thiolate anion) and a disulfide bond. The rate of thiol-disulfide exchange depends on the extent of ionization of the nucleophilic thiol, and therefore generally increases as the reaction pH increases until pK of the nucleophilic thiol group is exceeded. Even protein does not have free cysteine residues, disulfide bond scrambling may still occur, causing protein aggregation.
Amino acids, the components of proteins, are additionally subject to acid and base hydrolysis. Most peptide bonds are stable except those in -X-Asp-Y- sequence. During hydrolysis, aspartate forms succinimide intermediate, which is similar to the succinimide intermediate obtained during deamidation of Asn. In addition, formation of cyclic anhydride intermediate is also possible, especially when the C-flanking residue of aspartate is proline. In many cases, hydrolysis is a continuation after deamidation of asparagine residues.
Except for glycine, amino acids further have the potential of racemization. Aspartate-X peptide bonds can easily undergo a reversible isomerization between aspartate and iso-aspartate via a cyclic imide (succinimide) intermediate. The succinimide intermediate is usually not stable, and significant hydrolysis may occur within hours. Like deamidation, the rate of aspartate isomerization is strongly influenced by its location and mobility in a protein. The iso-aspartate formation is most likely to occur in relatively unstructured domains of intact proteins or domains susceptible to transient unfolding.
Formation of succinimide intermediates may precede deamidation of asparagine and isomerization of aspartate in proteins. In fact, formation of succinimide is the cause of iso-aspartate derivative formation in proteins. Asparagine deamidates via succinimide formation at neutral and alkaline conditions, but formation at Asp-Gly linkages in proteins may occur at an optimum pH of 4-5. The rate of succinimide formation is strongly influenced by neighboring groups of labile residues and by protein conformation.
Proteins may further form covalent dimers and polymers by non-disulfide pathways. For example, formaldehyde-mediated cross-linking causes significant aggregation of lyophilized tetanus and diphtheria toxoids during storage and thus raises concerns for storing formaldehyde-inactivated virus vaccine formulations in both liquid and solid forms.
Proteins may also be chemically transformed by deglycosylation, which renders the protein more sensitive to thermal denaturation, as one of the functions of carbohydrate moieties in proteins is to protect proteins from thermal and hydrolytic inactivation. The effect of glycosylation on the stability of proteins varies strongly from protein to protein.
Finally, sugars are often used as protein stabilizers in both liquid and solid formulations, however, reducing sugars may react with amino groups in proteins forming carbohydrate adducts, especially at high temperatures. This extremely complex browning pathway is known as Maillard reaction.
All of the above described influences—either simultaneously or separately—can occur when different types of stresses are applied to a protein, such as during isolation and purification of a protein, drying of a protein e.g. by lyophilisation, spray-drying, spray-freeze drying or foam drying, storage of a protein in solution or after drying as well as reconstitution after drying.
In particular during drying of a protein, considerable stress is applied. For example, during freeze-drying, pure crystalline ice forms from the liquid as it becomes frozen. Exposure of proteins to this ice-water interface can lead to denaturation, e.g. freezing damage. Further, removal of the hydration shell from proteins during drying in the absence of the appropriate stabilisers can cause an additional destabilisation of the protein structure. Furthermore, variations in pressure, pH value or ion concentration as well as concentration effects of additives, temperature variations and shear forces also affect the stability of a protein during drying. Also the influence of oxygen, surface effects or hydrolysis may inactivate or denature proteins. During storage of the dried protein, stress factors such as e.g. residual moisture, light exposure, oxidation as well as temperature during storage may further affect denaturation and inactivation of the dried proteins. All of the above aspects may result in problems in reconstitution of the proteins and loss of activity.
As it is more complicated to provide structural modifications of a protein of interest, commonly applied methods of protein stabilization are based on the addition of excipients. Excipients may reduce aggregation and may also retard certain chemical degradations in proteins. Their stabilizing effects are concentration- and protein-dependent, although high concentrations of excipients may not be necessarily more effective, and in some cases, can have negative effects. Frequently used protein stabilizers include sugars and polyols, amino acids, amines, salts, polymers and surfactants, each of which may exert different stabilizing effects.
Excipients are added to formulations for several reasons and some excipients may have more than one effect or purpose for being part of the formulation. In the choice of excipients, both physical and chemical stability have to be optimized. Excipients are often used to slow down or prevent the physical destabilization processes (protein aggregation). There are specific mechanisms of solvent-induced stabilization of proteins, which are specifically related to the excipients in the formulation. Stabilization is achieved by strengthening of the protein-stabilizing forces, by destabilization of the denatured state, or by direct binding of excipients to the protein.
The main function of stabilizers in pharmaceutical formulations thus is to protect the protein against the different types of stresses that are applied to a protein during isolation and purification of a protein, drying of a protein e.g. by lyophilization, spray-drying, spray-freeze drying or foam-drying, storage of a protein in solution or after drying as well as reconstitution after drying.
The structure of water surrounding a folded protein in solution is extremely important in maintaining the native structure of the protein. The presence of stabilizing excipients, such as sugars and amino acids, may stabilize the protein by a preferential exclusion of the excipients from the protein surface (the so-called preferential exclusion model) because of their thermodynamic unfavorable interactions with the peptide backbone, thus leading to the protein becoming preferentially hydrated, as more water molecules are found on the surface of the protein than in the bulk solution. This process is believed to act stabilizing upon exposure of proteins to typical stresses like isolation, purification, storage in solution, and preparation of liquid protein drug formulations. Protein stabilization by preferential exclusion depends on the concentration of the preferentially excluded excipient and requires sufficient amounts of water to be present at least locally.
During drying of proteins, the concentration of the excipient is increased, and the preferential exclusion effect is enhanced in the residual wet regions. Due to this process the protein remains hydrated in its native form until the residual water molecules are removed by further drying (freeze drying etc.). Upon removal of this residual water, hydrogen bonds are increasingly formed between the protein and the functional groups of the excipient, thereby replacing the missing interactions of the protein with the surrounding water molecules that constituted the hydration shell (water replacement, preferential interaction). Thus, excipients having lyoprotectant and/or cryoprotectant effects on the proteins are generally added to optimize protein stability during changes in water content. Examples of such excipients include e.g. sugars and polyols but also other excipients such as for example surfactants and amino acids. Commonly used excipients in the development of lyophilization formulations are discussed, for example, in Kofi Bedu-Addo 2004 (Pharmaceutical Technology, Lyophilization; 2004: 10-30).
The freeze-drying process yields a dried powder containing the protein in a glassy state, often including amorphous excipients and residual water. In the dried state, the rate of chemical degradation and unfolding is influenced by the mobility of the protein and the surrounding excipient molecules. Such mobility is greatly reduced in the glassy state of the dried protein, which can be further stabilized by the presence of amorphous excipients, such as e.g. trehalose. The residual moisture will depend on the solid state properties of the system, that is amorphous vs. crystalline, in combination with the chosen process conditions.
Excipients are also added to optimize a dry powder formulation. A dry powder needs to have certain characteristics to be useful. For example, a freeze-dried cake requires an acceptable appearance (as this is indicative of stability), should be rapidly dissolvable and blow-out of the formulation (i.e. foaming of the product after reconstitution) must be prevented. For this purpose, bulking agents such as sugars and polyols are generally selected, as they can also act as cryoprotectants and lyoprotectants. When selecting an appropriate excipient, the solid state properties are to be considered first. For example, mannitol will usually crystallize and thus lead to a cake with a good structural stability. However, mannitol can crystallize in three different polymorphic forms (α, β, δ) with different stabilities, and mannitol-hemi-hydrate, which may release its crystal water during storage and the solid state of mannitol depends on the freeze-drying conditions applied as well as the presence of other excipients. Sucrose usually remains amorphous on freeze-drying, which is desirable for protein stability, but it also increases the water content after primary drying and increases the danger for deliquescence and collapse of the final product.
Prevention of the direct interaction between proteins can also stabilize proteins, as these interactions most often lead to aggregation. For example arginine has previously been reported to bind strongly to some proteins while it has also been reported to be excluded from the surface of others.
Chemical instability can be minimized by the appropriate choice of preparation procedures, storage conditions, temperature, vials, or by addition of antioxidant such as for instance ascorbic acid. Ascorbic acid acts as an antioxidant to prevent oxidation of proteins. On the other hand, it also facilitates metal-catalyzed oxidation of proteins due to the reducing properties of ascorbate in the presence of metal ions and oxygen. This latter effect is usually avoided by co-adding chelating agents such as EDTA, DTPA, DFO.
Adsorption to interfaces is generally avoided by the addition of excipients that (ideally) are more surface-active than the protein itself. Mainly surfactants or other proteins are used to coat or adsorb competitively to the inner surface of the containers or adsorb to the surfaces created in the preparation of the delivery system. Surfactants may be classified as either ionic or non-ionic. Low concentrations of non-ionic surfactants are often sufficient to prevent or reduce protein surface adsorption or aggregation due to their relatively low critical micelle concentrations (CMC). Examples of generally employed non-ionic surfactants include poloxamer (Pluronic F-68) and polyoxyethyleneglycol dodecyl ether (Brij35), polysorbate 80, 20, Human Serum Albumine etc. Some of these surfactants, particularly the polysorbates, may be contaminated with alkyl peroxides arising from the ether linkage incorporated in their structures, which is disadvantageous as it can accelerate the oxidation of proteins. Polymers and dextran can also be used to protect against surface adsorption, although only large PEGs are reported to have a stabilizing effect on proteins, while small PEGs appear to induce unfolding. The chosen concentration of surfactant depends on the effect that needs to be avoided, but typically it is just above the CMC value, where a monolayer of the surfactant is present at the interface. Examples of generally employed ionic surfactants include Cetyltrimethylammonium chloride (CTAC) and Cetyltrimethylammonium bromid (CTAB).
During the process of preparing formulations, for example during the drying process, changes in the microclimate pH can occur, owing to changes in the proteins microenvironment. In drying process one component will stay in solution for a longer period than others, which can lead to a pH shift of more than three pH units. Smaller pH changes are also encountered during temperature changes such as lyophilization, spray drying, storage, etc. as the pH is dependent on temperature. However, proteins are usually only stable over a narrow pH range. Thus, an important step early in developing the appropriate formulation is to study the pH stability, especially in the range between pH 3-10.
Maintenance of the pH is achieved by employing the appropriate buffer system. Unfortunately, however, there are no general rules for specific buffer selection. Usually, specific excipients for avoiding pH changes are not added, but instead very low concentrations of buffer should be used, if it is possible. When choosing a buffer, it has to be kept in mind that the buffer system may affect the chemical stability of the formulation, as the pH value appears to be the major controlling variable in deamidation reactions and that aggregation rates are influenced by the choice of buffer.
Salts are used frequently for the adjustment of pH and tonicity. During freezing the pure solvent (water) freezes first, leading to an increase of the salt concentration in the remaining liquid phase (freeze concentrated phase), thereby increasing the ionic strength.
Finally, in some cases, an isotonic formulation might be required either due to the stability requirements of the bulk solution or the requirements for the route of administration. Excipients such as mannitol, sucrose, glycine, glycerol, and sodium chloride are good tonicity adjusters. Tonicity modifiers also can be included in the diluent rather than the formulation.